Various gene transfer techniques have been developed in order to introduce genes of interest to cells, tissues, or individuals and express the genes. One of such techniques is viral vectors based on the infectious or replicating ability of viruses. Various vectors derived from retrovirus, adenovirus, adeno-associated virus, and the like are known. Among them, adeno-associated virus vectors have been expected as gene transfer carriers useful in gene therapy in recent years.
The adeno-associated virus (AAV; hereinafter, referred to as “AAV” in the present specification) is a non-pathogenic virus that belongs to the family Parvoviridae. This virus lacks self-renewal ability and therefore cannot autonomously grow. Since its growth requires coinfection by adenovirus or herpesvirus, AAV is low infective. AAV also has the property of low immunogenicity in hosts. Owing to such features, AAV is advantageously highly safe as a gene transfer carrier. In addition, AAV is capable of infecting various cells because of its wide host range. Moreover, vectors derived from each of AAV serotypes 1 to 9 have also been developed (Non Patent Literature 1) and therefore permit gene expression in particular cells (e.g., nerve cells, muscle cells, and hepatic cells), tissues, and organs by the application of the specificity of the target cells to be infected for each serotype.
All of the conventional viral vectors including AAV vectors, however, have presented various problems against recipient individuals, such as in vivo transient gene expression, possible contamination by wild-type viruses, time required for gene expression and manifestation of the action thereof, impossible incorporation of large genes, and the need of a mechanism for controlling expression when side reaction appears.
In order to solve these problems, Samulski et al. have proposed expressing capsids with AAV capsid expression plasmids and using them as drug delivery carriers (Non Patent Literature 2). Vacant capsids containing no viral genome therein, i.e., empty capsids, have viral early infection activities such as specific recognition of target cells, adsorption, penetration, and uncoating, but lack a viral growth activity because of having no virus-derived gene. Thus, the empty capsids can serve as ideal drug delivery system (DDS) carriers that have both the target cell-specific delivery of drugs and safety in recipient individuals. Hence, the technique of introducing drugs of interest to the inside of the capsids is very important for using the empty capsids as DDS carriers.
Samulski et al. have reported an introduction method which involves temporarily denaturing an empty capsid using urea, heat, or pH conditions to allow the capsid to take up a drug, and then reconstituting the resulting capsid (Non Patent Literature 2). Upon action of a denaturant such as urea, however, the capsid disadvantageously loses its early infection activities and is thus no longer able to function as a delivery carrier. After all, the technique of introducing drugs as substances to be delivered into empty capsids with the early infection activities of the capsids kept has not yet been established even though 7 or more years have already passed from the proposal. For example, Patent Literature 1 suggests the usefulness of empty AAV particles as delivery carriers, but makes no mention about specific examples of empty capsids used as delivery carriers. Hence, those skilled in the art have shared a common understanding that the technique proposed by Samulski is impossible to realize.